Bottom Line:
Injection of K5-NP significantly reduced retinal vascular leakage and attenuated retinal neovascularization, when compared with the contralateral eyes injected with Control-NP in oxygen-induced retinopathy rats.No toxicities of K5-NP were detected to retinal structure and function.K5-NP mediates efficient and sustained K5 expression in the retina and has therapeutic potential for diabetic retinopathy.

Objective: The aim of the study is to evaluate the effect of nanoparticle-mediated gene delivery of angiogenic inhibitors on retinal inflammation, vascular leakage, and neovascularization in diabetic retinopathy.

Research design and methods: An expression plasmid of plasminogen kringle 5 (K5), a natural angiogenic inhibitor, was encapsulated with poly(lactide-coglycolide) to form K5 nanoparticles (K5-NP). Expression of K5 was determined by Western blot analysis and immunohistochemistry, and retinal vascular leakage was measured by permeability assay. Retinal neovascularization was evaluated using fluorescein-angiography and counting preretinal vascular cells in rats with oxygen-induced retinopathy. Effects of K5-NP on retinal inflammation were evaluated in streptozotocin-induced diabetic rats by leukostasis assay and Western blot analysis of intracellular adhesion molecule and vascular endothelial growth factor. Possible toxicities of K5-NP were evaluated using histology examination, retinal thickness measurement, and electroretinogram recording.

Results: K5-NP mediated efficient expression of K5 and specifically inhibited growth of endothelial cells. An intravitreal injection of K5-NP resulted in high-level expression of K5 in the inner retina of rats during the 4 weeks they were analyzed. Injection of K5-NP significantly reduced retinal vascular leakage and attenuated retinal neovascularization, when compared with the contralateral eyes injected with Control-NP in oxygen-induced retinopathy rats. K5-NP attenuated vascular endothelial growth factor and intracellular adhesion molecule-1 overexpression and reduced leukostasis and vascular leakage for at least 4 weeks after a single injection in the retina of streptozotocin-induced diabetic rats. No toxicities of K5-NP were detected to retinal structure and function.

Conclusions: K5-NP mediates efficient and sustained K5 expression in the retina and has therapeutic potential for diabetic retinopathy.

Figure 5: Effect of K5-NP on retinal neovascularization in OIR rats. K5-NP was injected into the vitreous of the right eyes (8.8 μg/eye) and the same amount of control-NP into the contralateral eyes of seven OIR rats at P12. Retinal vasculature was examined using fluorescein angiography at P18 as described in methods. A and C: Representative retinal angiographs from the eyes injected with control-NP; B and D are representative angiographs from the K5-NP–injected eyes (40× in A and B; 100× in C and D). Scale bar: A and B, 100 μm; C and D, 40 μm. E: Retinal neovascularization was quantified by measuring the neovascular area in the retina and expressed as percent of the total retina area (mean ± SD, n = 7). The difference of the neovascular area was compared with the contralateral eyes using paired Student's t test. (A high-quality digital representation of this figure is available in the online issue.)

Mentions:
To evaluate the effect of K5-NP on retinal neovascularization, K5-NP was injected intravitreally into the right eyes (8.8 μg/eye) of the OIR rats at P12 and Control-NP into the left eyes. The retinal vasculature was visualized and examined by fluorescein retinal angiography at P18. Retinal angiographs on retinal flat mounts showed that the eyes injected with Control-NP developed severe retinal neovascularization in the OIR rats (Fig. 5A and C). In contrast, a single K5-NP injection ameliorated the retinal neovascularization in the same rats by decreasing neovascular areas and nonperfusion areas in the retina (Fig. 5B and D). The neovascularization was semi-quantified by measuring the ratio of the neovascular area to the total retina area, which showed that the eyes injected with K5-NP have significantly decreased retinal neovascular areas in the OIR rats, compared with those injected with Control-NP (Fig. 5E).

Figure 5: Effect of K5-NP on retinal neovascularization in OIR rats. K5-NP was injected into the vitreous of the right eyes (8.8 μg/eye) and the same amount of control-NP into the contralateral eyes of seven OIR rats at P12. Retinal vasculature was examined using fluorescein angiography at P18 as described in methods. A and C: Representative retinal angiographs from the eyes injected with control-NP; B and D are representative angiographs from the K5-NP–injected eyes (40× in A and B; 100× in C and D). Scale bar: A and B, 100 μm; C and D, 40 μm. E: Retinal neovascularization was quantified by measuring the neovascular area in the retina and expressed as percent of the total retina area (mean ± SD, n = 7). The difference of the neovascular area was compared with the contralateral eyes using paired Student's t test. (A high-quality digital representation of this figure is available in the online issue.)

Mentions:
To evaluate the effect of K5-NP on retinal neovascularization, K5-NP was injected intravitreally into the right eyes (8.8 μg/eye) of the OIR rats at P12 and Control-NP into the left eyes. The retinal vasculature was visualized and examined by fluorescein retinal angiography at P18. Retinal angiographs on retinal flat mounts showed that the eyes injected with Control-NP developed severe retinal neovascularization in the OIR rats (Fig. 5A and C). In contrast, a single K5-NP injection ameliorated the retinal neovascularization in the same rats by decreasing neovascular areas and nonperfusion areas in the retina (Fig. 5B and D). The neovascularization was semi-quantified by measuring the ratio of the neovascular area to the total retina area, which showed that the eyes injected with K5-NP have significantly decreased retinal neovascular areas in the OIR rats, compared with those injected with Control-NP (Fig. 5E).

Bottom Line:
Injection of K5-NP significantly reduced retinal vascular leakage and attenuated retinal neovascularization, when compared with the contralateral eyes injected with Control-NP in oxygen-induced retinopathy rats.No toxicities of K5-NP were detected to retinal structure and function.K5-NP mediates efficient and sustained K5 expression in the retina and has therapeutic potential for diabetic retinopathy.

Objective: The aim of the study is to evaluate the effect of nanoparticle-mediated gene delivery of angiogenic inhibitors on retinal inflammation, vascular leakage, and neovascularization in diabetic retinopathy.

Research design and methods: An expression plasmid of plasminogen kringle 5 (K5), a natural angiogenic inhibitor, was encapsulated with poly(lactide-coglycolide) to form K5 nanoparticles (K5-NP). Expression of K5 was determined by Western blot analysis and immunohistochemistry, and retinal vascular leakage was measured by permeability assay. Retinal neovascularization was evaluated using fluorescein-angiography and counting preretinal vascular cells in rats with oxygen-induced retinopathy. Effects of K5-NP on retinal inflammation were evaluated in streptozotocin-induced diabetic rats by leukostasis assay and Western blot analysis of intracellular adhesion molecule and vascular endothelial growth factor. Possible toxicities of K5-NP were evaluated using histology examination, retinal thickness measurement, and electroretinogram recording.

Results: K5-NP mediated efficient expression of K5 and specifically inhibited growth of endothelial cells. An intravitreal injection of K5-NP resulted in high-level expression of K5 in the inner retina of rats during the 4 weeks they were analyzed. Injection of K5-NP significantly reduced retinal vascular leakage and attenuated retinal neovascularization, when compared with the contralateral eyes injected with Control-NP in oxygen-induced retinopathy rats. K5-NP attenuated vascular endothelial growth factor and intracellular adhesion molecule-1 overexpression and reduced leukostasis and vascular leakage for at least 4 weeks after a single injection in the retina of streptozotocin-induced diabetic rats. No toxicities of K5-NP were detected to retinal structure and function.

Conclusions: K5-NP mediates efficient and sustained K5 expression in the retina and has therapeutic potential for diabetic retinopathy.